The ability to cryopreserve oocytes, embryos, sperm and other similar biological specimens is critical to the widespread application of assisted reproductive technologies. However, due to the large volume of the cells and the high chilling sensitivity of oocytes and early embryos, cryopreservation techniques are not well developed in most species.
Traditionally, embryos are cryopreserved using “slow freezing techniques”. Low concentrations of cryoprotectants and slow controlled rates of cooling usually in the range of 0.1–0.3° C./min. slowly dehydrate the cell during freezing to prevent intracellular crystallization. Because of this, cryopreservation of oocytes, embryos and other developmental cells using such procedures results in a reduced ability to both establish and maintain pregnancy following transfer. Oocytes are particularly susceptible to cryopreservation damage because of disruption of the metaphase spindle microtubule integrity during cooling.
Alternative prior cryopreservation methods have relied on vitrification with high concentrations of cryoprotectants, which when rapidly cooled result in a glass-like state. However, a disadvantage of this vitrification technique is that the cryoprotectants are very toxic to oocytes, embryos and other delicate developmental cells. Cryoprotectant toxicity can be minimized by increasing the cooling rate, which has been accomplished by plunging oocytes held on electron microscopy grids, or within thinly walled straws (known as open pulled straw) directly into liquid nitrogen. However, both of these procedures are cumbersome and recovery of embryos is problematic.
Therefore a need remains for a method for the vitrification of a biological specimen which is able to maximize the cooling rate of the cells of the specimen; maintain viability of the specimen during vitrification and subsequent thawing; prevent mechanical stress to the specimen; and provide ease of manipulations during cryopreservation and recovery.